Pre-visualization of the normalized count data before any differential analysis

Table of content

1. Sample clustering
   1. With all genes
   2. Without X/Y genes
2. PCA on the normalized counts
   1. With all factors
   2. SPF vs GF
3. Gene co-expression analysis
   1. Extract modules of co-expressed genes
   2. Relationship between modules and samples
   3. Genes in modules
      1. Heatmaps with Z-scores
      2. Sinaplots of the Z-scores per groups
      3. Median of counts over ages
   4. Enrichment analysis in modules
   5. Network visualization
   6. Functional gene network

Load

Libraries and functions

Warning message in rsqlite_fetch(res@ptr, n = n):
“Don't need to call dbFetch() for statements, only for queries”

==========================================================================
*
*  Package WGCNA 1.63 loaded.
*
*    Important note: It appears that your system supports multi-threading,
*    but it is not enabled within WGCNA in R. 
*    To allow multi-threading within WGCNA with all available cores, use 
*
*          allowWGCNAThreads()
*
*    within R. Use disableWGCNAThreads() to disable threading if necessary.
*    Alternatively, set the following environment variable on your system:
*
*          ALLOW_WGCNA_THREADS=<number_of_processors>
*
*    for example 
*
*          ALLOW_WGCNA_THREADS=4
*
*    To set the environment variable in linux bash shell, type 
*
*           export ALLOW_WGCNA_THREADS=4
*
*     before running R. Other operating systems or shells will
*     have a similar command to achieve the same aim.
*
==========================================================================


Allowing multi-threading with up to 4 threads.
[1] "preparing gene to GO mapping data..."
[1] "preparing IC data..."
[1] "preparing gene to GO mapping data..."
[1] "preparing IC data..."
[1] "preparing gene to GO mapping data..."
[1] "preparing IC data..."

Data

	GF_104w_F_1_2	GF_104w_F_2_2	GF_104w_F_3_2	GF_104w_M_1_2	GF_104w_M_2_2	GF_52w_M_1_2	GF_52w_M_2_2	GF_52w_M_3_2	GF_52w_M_4_2	GF_8w_M_1_2	⋯	SPF_8w_F_3_2	SPF_8w_F_4_2	GF_8w_F_1_2	GF_8w_F_2_2	GF_8w_F_3_2	GF_8w_F_4_2	GF_8w_F_5_2	GF_104w_M_3_2	GF_104w_M_5_2	GF_104w_M_4_2
0610005C13Rik	0.00000	0.00000	0.00000	1.762244	1.255776	0.8428813	4.933784	5.600068	7.223366	8.467973	⋯	2.104621	2.598973	18.22456	0.0000	2.02195	5.263542	0.0000	1.256186	1.048314	1.510221
0610007P14Rik	863.76231	747.10939	907.37337	882.884096	990.178987	509.1002974	529.888431	514.086266	737.505715	603.645538	⋯	741.878817	907.041617	897.29135	681.3924	611.63981	592.674855	807.4643	494.937435	597.539204	749.069592
0610009B22Rik	1015.87139	1165.23596	1177.13303	1220.353766	1554.022190	1040.1155082	875.253330	1015.852381	876.916688	948.413030	⋯	1160.698348	1294.288611	1038.79966	1019.3717	1140.37968	959.017393	1091.5484	692.158697	809.298711	788.335337
0610009L18Rik	28.06775	36.08199	18.16563	20.265803	38.301153	32.0294889	26.642435	22.400273	4.334020	61.695235	⋯	45.249346	102.659438	48.24147	19.5615	11.12072	45.266463	24.3135	25.123728	23.062917	39.265745
0610009O20Rik	216.39328	278.04355	265.21824	225.567194	271.247510	300.0657382	349.311927	395.364819	309.160084	360.493728	⋯	251.502181	227.410148	231.55906	158.6655	270.94127	304.232741	268.7282	337.914137	367.958352	397.188110
0610010F05Rik	409.24587	459.51473	287.92528	277.553384	303.269785	392.7826798	405.557067	388.644737	357.556640	343.557781	⋯	384.093288	296.282935	541.37650	399.9241	406.41191	528.459639	321.1942	517.548790	566.089772	312.615737

Sample clustering

With all genes

Without X/Y genes

PCA on the normalized counts

With all factors

SPF vs GF

Order:

• M, 8w
• M, 52w
• M, 104w
• F, 8w
• F, 52w
• F, 104w

Gene co-expression analysis

Extract modules of co-expressed genes

1. 19694
2. 61

1. 10291
2. 61

pickSoftThreshold: will use block size 4347.
 pickSoftThreshold: calculating connectivity for given powers...
   ..working on genes 1 through 4347 of 10291
   ..working on genes 4348 through 8694 of 10291
   ..working on genes 8695 through 10291 of 10291
   Power SFT.R.sq  slope truncated.R.sq  mean.k. median.k. max.k.
1      1    0.227  1.310          0.952 1030.000  1.03e+03 1570.0
2      2    0.232 -0.842          0.911  328.000  3.12e+02  758.0
3      3    0.590 -1.550          0.954  132.000  1.15e+02  446.0
4      4    0.729 -1.880          0.974   61.300  4.74e+01  292.0
5      5    0.771 -2.080          0.972   31.700  2.12e+01  205.0
6      6    0.812 -2.130          0.981   17.800  1.01e+01  150.0
7      7    0.802 -2.220          0.974   10.600  5.05e+00  114.0
8      8    0.831 -2.190          0.983    6.630  2.61e+00   88.7
9      9    0.852 -2.140          0.988    4.330  1.40e+00   70.5
10    10    0.867 -2.110          0.991    2.920  7.74e-01   56.8
11    12    0.872 -2.060          0.983    1.440  2.55e-01   38.4
12    14    0.877 -2.010          0.977    0.779  9.07e-02   26.9
13    16    0.893 -1.940          0.982    0.449  3.40e-02   19.4
14    18    0.906 -1.860          0.985    0.273  1.33e-02   14.2
15    20    0.929 -1.780          0.993    0.173  5.31e-03   10.7

Power	SFT.R.sq	slope	truncated.R.sq	mean.k.	median.k.	max.k.
1	0.2273633	1.3069408	0.9522773	1032.2103062	1.031743e+03	1574.40467
2	0.2318240	-0.8419065	0.9109768	328.3750082	3.124206e+02	758.22758
3	0.5898524	-1.5468341	0.9541521	131.7106717	1.147058e+02	446.45478
4	0.7293365	-1.8760203	0.9740987	61.3157190	4.736691e+01	292.33277
5	0.7706350	-2.0775848	0.9724795	31.7270126	2.117472e+01	204.76919
6	0.8116126	-2.1271367	0.9807292	17.7731786	1.008260e+01	150.23963
7	0.8022879	-2.2224796	0.9735875	10.5925115	5.050971e+00	114.00564
8	0.8307511	-2.1879600	0.9827800	6.6345171	2.608895e+00	88.74112
9	0.8519550	-2.1423593	0.9884310	4.3282040	1.397797e+00	70.46121
10	0.8665930	-2.1052573	0.9914710	2.9212252	7.738615e-01	56.84323
12	0.8724663	-2.0611029	0.9830544	1.4447620	2.552006e-01	38.38129
14	0.8766131	-2.0101797	0.9767692	0.7788890	9.066889e-02	26.89092
16	0.8933757	-1.9356365	0.9817629	0.4488075	3.402853e-02	19.36386
18	0.9058564	-1.8638594	0.9845667	0.2726832	1.328237e-02	14.24265
20	0.9285263	-1.7822036	0.9931630	0.1729982	5.314736e-03	10.65525

	Power	mean.k.
6	6	17.7731786
7	7	10.5925115
8	8	6.6345171
9	9	4.3282040
10	10	2.9212252
11	12	1.4447620
12	14	0.7788890
13	16	0.4488075
14	18	0.2726832
15	20	0.1729982

Parameters for network construction

• Soft-thresholding power parameter: 5 (R2 > 0.80 and mean connectivity highest as possible (Ref: https://www.degruyter.com/view/j/sagmb.2005.4.issue-1/sagmb.2005.4.1.1128/sagmb.2005.4.1.1128.pdf))
• Minimum module size: 50
• Height for module merging: 0.35 (below)
• Biweight Midcorrelation

 Calculating module eigengenes block-wise from all genes
   Flagging genes and samples with too many missing values...
    ..step 1
 ..Working on block 1 .
    TOM calculation: adjacency..
    ..will use 4 parallel threads.
     Fraction of slow calculations: 0.000000
    ..connectivity..
    ..matrix multiplication (system BLAS)..
    ..normalization..
    ..done.
   ..saving TOM for block 1 into file norm_genes_TOM-block.1.RData
 ....clustering..
 ....detecting modules..
 ....calculating module eigengenes..
 ....checking kME in modules..
     ..removing 1 genes from module 1 because their KME is too low.
     ..removing 3 genes from module 9 because their KME is too low.
     ..removing 1 genes from module 12 because their KME is too low.
     ..removing 1 genes from module 13 because their KME is too low.
 ..merging modules that are too close..
     mergeCloseModules: Merging modules whose distance is less than 0.35
       Calculating new MEs...

 ME0  ME1  ME2  ME3  ME4  ME5  ME6  ME7  ME8  ME9 ME10 ME11 
4663 1239 1082  630  595  552  476  367  315  178  139   55 

11

• 11 modules with between 55 and 1239 genes
• 4663 genes not assigned to a module

5628

0

'grey'

1

'turquoise'

2

'blue'

3

'brown'

4

'yellow'

5

'green'

6

'red'

7

'black'

8

'pink'

9

'magenta'

10

'purple'

11

'greenyellow'

Relationship between modules and samples

Genes in modules

[1] "0: 147 - 3.15247694617199%"
[1] "1: 36 - 2.90556900726392%"
[1] "2: 53 - 4.89833641404806%"
[1] "3: 18 - 2.85714285714286%"
[1] "4: 13 - 2.18487394957983%"
[1] "5: 19 - 3.44202898550725%"
[1] "6: 9 - 1.89075630252101%"
[1] "7: 7 - 1.90735694822888%"
[1] "8: 9 - 2.85714285714286%"
[1] "9: 2 - 1.12359550561798%"
[1] "10: 1 - 0.719424460431655%"
[1] "11: 1 - 1.81818181818182%"

Heatmaps with Z-scores

Sinaplots of the Z-scores per groups

The mean of the Z-score over the samples in the group is plot for each gene

Median of counts over ages

Enrichment analysis in modules

[1] "0"

Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Warning message in pcls(G):
“initial point very close to some inequality constraints”Fetching GO annotations...
For 1495 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns

        category over_represented_pvalue under_represented_pvalue numDEInCat
9622  GO:0034641            1.930318e-34                        1       1689
11864 GO:0044237            1.819262e-33                        1       2515
9636  GO:0034660            1.511191e-32                        1        212
11881 GO:0044260            2.808359e-32                        1       2109
2983  GO:0006139            6.603118e-32                        1       1518
9532  GO:0034470            7.640556e-32                        1        169
      numInCat                                             term ontology
9622      5285     cellular nitrogen compound metabolic process       BP
11864     8386                       cellular metabolic process       BP
9636       405                          ncRNA metabolic process       BP
11881     6844         cellular macromolecule metabolic process       BP
2983      4672 nucleobase-containing compound metabolic process       BP
9532       301                                 ncRNA processing       BP
[1] "1"

Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Warning message in pcls(G):
“initial point very close to some inequality constraints”Fetching GO annotations...
For 1495 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns

        category over_represented_pvalue under_represented_pvalue numDEInCat
17661 GO:0090304            3.630391e-20                        1        450
11881 GO:0044260            1.095788e-18                        1        644
5710  GO:0016070            1.118447e-17                        1        406
2983  GO:0006139            9.921890e-17                        1        468
12852 GO:0046483            4.487238e-16                        1        471
11364 GO:0043170            1.928903e-15                        1        667
      numInCat                                             term ontology
17661     4172                   nucleic acid metabolic process       BP
11881     6844         cellular macromolecule metabolic process       BP
5710      3729                            RNA metabolic process       BP
2983      4672 nucleobase-containing compound metabolic process       BP
12852     4765                    heterocycle metabolic process       BP
11364     7455                  macromolecule metabolic process       BP
[1] "2"

Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Warning message in pcls(G):
“initial point very close to some inequality constraints”Fetching GO annotations...
For 1495 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns

        category over_represented_pvalue under_represented_pvalue numDEInCat
12259 GO:0045184            9.868354e-13                        1        181
11879 GO:0044257            1.106652e-12                        1         84
14123 GO:0051234            2.182943e-12                        1        345
5312  GO:0015031            2.460062e-12                        1        167
13314 GO:0048193            2.934615e-12                        1         40
3431  GO:0006810            4.433883e-12                        1        333
      numInCat                                  term ontology
12259     1765 establishment of protein localization       BP
11879      604    cellular protein catabolic process       BP
14123     4062         establishment of localization       BP
5312      1606                     protein transport       BP
13314      185               Golgi vesicle transport       BP
3431      3915                             transport       BP
[1] "3"

Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Warning message in pcls(G):
“initial point very close to some inequality constraints”Fetching GO annotations...
For 1495 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns

        category over_represented_pvalue under_represented_pvalue numDEInCat
17661 GO:0090304            8.971982e-19                        1        245
5710  GO:0016070            1.893249e-17                        1        223
14136 GO:0051252            2.138832e-15                        1        190
2983  GO:0006139            5.935538e-15                        1        251
12852 GO:0046483            7.973750e-15                        1        254
14083 GO:0051171            1.403879e-14                        1        210
      numInCat                                              term ontology
17661     4172                    nucleic acid metabolic process       BP
5710      3729                             RNA metabolic process       BP
14136     3046               regulation of RNA metabolic process       BP
2983      4672  nucleobase-containing compound metabolic process       BP
12852     4765                     heterocycle metabolic process       BP
14083     3601 regulation of nitrogen compound metabolic process       BP
[1] "4"

Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Fetching GO annotations...
For 1495 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns

        category over_represented_pvalue under_represented_pvalue numDEInCat
11864 GO:0044237            3.108584e-18                        1        368
16807 GO:0071704            1.641624e-16                        1        377
11881 GO:0044260            1.908849e-16                        1        307
3934  GO:0008152            1.988270e-16                        1        390
11865 GO:0044238            2.158860e-16                        1        361
11364 GO:0043170            2.641483e-16                        1        328
      numInCat                                     term ontology
11864     8386               cellular metabolic process       BP
16807     8831      organic substance metabolic process       BP
11881     6844 cellular macromolecule metabolic process       BP
3934      9217                        metabolic process       BP
11865     8362                primary metabolic process       BP
11364     7455          macromolecule metabolic process       BP
[1] "5"

Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Fetching GO annotations...
For 1495 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns

        category over_represented_pvalue under_represented_pvalue numDEInCat
3553  GO:0007005            4.130830e-20                        1         70
3934  GO:0008152            1.159491e-17                        1        342
14836 GO:0055114            5.773753e-16                        1         76
11864 GO:0044237            7.659119e-16                        1        309
3431  GO:0006810            8.277589e-16                        1        167
14123 GO:0051234            1.090689e-14                        1        167
      numInCat                          term ontology
3553       611    mitochondrion organization       BP
3934      9217             metabolic process       BP
14836      836   oxidation-reduction process       BP
11864     8386    cellular metabolic process       BP
3431      3915                     transport       BP
14123     4062 establishment of localization       BP
[1] "6"

Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Fetching GO annotations...
For 1495 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns

        category over_represented_pvalue under_represented_pvalue numDEInCat
12058 GO:0044710            6.653859e-23                        1        174
4336  GO:0009056            3.116046e-13                        1         88
12060 GO:0044712            4.296236e-13                        1         52
18752 GO:1901575            4.041497e-12                        1         83
3934  GO:0008152            1.327891e-11                        1        306
2882  GO:0005975            1.537646e-11                        1         41
      numInCat                                term ontology
12058     3280   single-organism metabolic process       BP
4336      1533                   catabolic process       BP
12060      635   single-organism catabolic process       BP
18752     1478 organic substance catabolic process       BP
3934      9217                   metabolic process       BP
2882       489      carbohydrate metabolic process       BP
[1] "7"

Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Warning message in pcls(G):
“initial point very close to some inequality constraints”Fetching GO annotations...
For 1495 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns

        category over_represented_pvalue under_represented_pvalue numDEInCat
13833 GO:0050794            2.673494e-09                        1        248
6626  GO:0019222            1.600242e-08                        1        167
5710  GO:0016070            2.997303e-08                        1        129
7965  GO:0031323            3.075312e-08                        1        159
13829 GO:0050789            4.088212e-08                        1        253
11881 GO:0044260            4.439255e-08                        1        201
      numInCat                                     term ontology
13833     8793           regulation of cellular process       BP
6626      5208          regulation of metabolic process       BP
5710      3729                    RNA metabolic process       BP
7965      4897 regulation of cellular metabolic process       BP
13829     9278         regulation of biological process       BP
11881     6844 cellular macromolecule metabolic process       BP
[1] "8"

Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Fetching GO annotations...
For 1495 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns

        category over_represented_pvalue under_represented_pvalue numDEInCat
3523  GO:0006955            2.985898e-25                        1         70
1046  GO:0002376            3.655761e-25                        1        100
3520  GO:0006952            1.989081e-22                        1         72
12206 GO:0045087            7.941872e-22                        1         47
11393 GO:0043207            2.651971e-17                        1         49
14391 GO:0051707            2.651971e-17                        1         49
      numInCat                                 term ontology
3523       982                      immune response       BP
1046      1975                immune system process       BP
3520      1156                     defense response       BP
12206      500               innate immune response       BP
11393      686 response to external biotic stimulus       BP
14391      686           response to other organism       BP
[1] "9"

Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Warning message in pcls(G):
“initial point very close to some inequality constraints”Fetching GO annotations...
For 1495 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns

        category over_represented_pvalue under_represented_pvalue numDEInCat
1211  GO:0002682            2.613030e-07                0.9999999         30
1213  GO:0002684            2.877916e-07                0.9999999         23
952   GO:0002221            1.182405e-06                0.9999999          9
7990  GO:0031349            1.266030e-06                0.9999998         13
1267  GO:0002758            1.395896e-06                0.9999999          9
13445 GO:0048518            1.634916e-06                0.9999992         77
      numInCat                                                  term ontology
1211      1088                   regulation of immune system process       BP
1213       676          positive regulation of immune system process       BP
952        104        pattern recognition receptor signaling pathway       BP
7990       252               positive regulation of defense response       BP
1267       106 innate immune response-activating signal transduction       BP
13445     4751             positive regulation of biological process       BP
[1] "10"

Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Warning message in pcls(G):
“initial point very close to some inequality constraints”Fetching GO annotations...
For 1495 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns

[1] category                 over_represented_pvalue  under_represented_pvalue
[4] numDEInCat               numInCat                 term                    
[7] ontology                
<0 rows> (or 0-length row.names)
[1] "11"

Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Warning message in pcls(G):
“initial point very close to some inequality constraints”Fetching GO annotations...
For 1495 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns

[1] category                 over_represented_pvalue  under_represented_pvalue
[4] numDEInCat               numInCat                 term                    
[7] ontology                
<0 rows> (or 0-length row.names)

Network visualization

TOM calculation: adjacency..
..will use 4 parallel threads.
 Fraction of slow calculations: 0.000000
..connectivity..
..matrix multiplication (system BLAS)..
..normalization..
..done.

	0610007P14Rik	0610009B22Rik	0610009O20Rik	0610010K14Rik	0610030E20Rik	0610037L13Rik	Fmc1	Card19	1110008F13Rik	1110008P14Rik	⋯	Zswim7	Zswim8	Zufsp	Zwint	Zxda	Zyg11b	Zyx	Zzef1	Zzz3	Hikeshi
0610007P14Rik	1	1	0	0	0	0	0	0	1	0	⋯	0	1	0	0	0	0	0	0	0	0
0610009B22Rik	1	1	1	0	0	0	0	0	0	0	⋯	0	1	0	0	0	0	0	0	0	1
0610009O20Rik	0	1	1	0	0	0	0	1	0	0	⋯	0	1	0	0	1	0	0	0	0	0
0610010K14Rik	0	0	0	1	0	0	0	1	0	0	⋯	1	0	0	0	0	1	1	0	0	1
0610030E20Rik	0	0	0	0	1	1	0	0	1	0	⋯	0	1	1	1	0	1	0	1	1	0
0610037L13Rik	0	0	0	0	1	1	0	0	0	0	⋯	0	1	1	0	0	0	0	1	1	0

0610007P14Rik

'5'

0610009B22Rik

'2'

0610009O20Rik

'7'

0610010K14Rik

'5'

0610030E20Rik

'1'

0610037L13Rik

'3'

5

'green'

2

'blue'

7

'black'

5

'green'

1

'turquoise'

3

'brown'